Europa,schesP_
J
European Patent Office
_„.
Office
europeen des brevets
EUROPEAN
©
©
MM II M M M MINI I MM II II I II
© Publication number:
PATENT
Date of publication of patent specification: 25.01.95
_ _ _ _
0 4^ 9 3 2 6 5_ B 1
SPECIFICATION
Int. CI.6: A23L 1/0532, A23L 1 / 3 0 8 ,
C08B 3 7 / 0 4
©
© Application number: 91403558.9
@ Date of filing: 27.12.91
© Algin-containing food and beverage.
®
©
Priority: 28.12.90 JP 416688/90
01.08.91 JP 193270/91
@ Date of publication of application:
01.07.92 Bulletin 92/27
©
Publication of the grant of the patent:
25.01.95 Bulletin 95/04
©
Designated Contracting States:
DE FR GB
©
References cited:
GB-A- 760 030
US-A- 3 423 397
PATENT ABSTRACTS OF JAPAN vol. 5, no.
200 (C-84)(872) 18 December 1981
PATENT ABSTRACTS OF JAPAN vol. 15, no.
80 (C-810)(4608) 25 February 1991
00
m
CO
CM
00
Oi
Proprietor: SUMITOMO METAL INDUSTRIES,
LTD.
5-33, Kltahama 4-chome
Chuo-ku
Osaka-Shi
Osaka 541 (JP)
@ Inventor: Iwata, Kazuyukl
406 Furontla-Enlwa,
220 Sakura-cho
Enlwa-shl,
Hokkaido (JP)
Inventor: Watanabe, Kazuhlro
304 Roman-Coto,
161-1 Alol-cho
Enlwa-shl,
Hokkaido (JP)
Inventor: Klmura, Yoshlyukl, 308 GuranDorumu-Fushlml-Klty
33-1 Fukenotsubo-cho
Fushlml-ku,
Kyoto-shl,
Kyoto-fu (JP)
Inventor: Okuda, Hlromlchl
1174-17 Takanoko
Matsuyama-shl,
Ehlme-ken (JP)
Note: Within nine months from the publication of the mention of the grant of the European patent, any person
may give notice to the European Patent Office of opposition to the European patent granted. Notice of opposition
shall be filed in a written reasoned statement. It shall not be deemed to have been filed until the opposition fee
has been paid (Art. 99(1) European patent convention).
Rank Xerox (UK) Business Services
(3. 10/3.09/3.3.3)
EP 0 493 265 B1
CHEMICAL ABSTRACTS, vol. 110, no. 15, 10
April 1989, Columbus, Ohio, US;abstract no.
135609R, T.D.BRADLEY ET EL: 'The determination of the kinetics of polysaccharide
thermal degradation using high temperature
viscosity measurements' page 753 ;column
R;
0
2
Representative: Schrlmpf, Robert et al
Cabinet Reglmbeau
26, Avenue Kleber
F-75116 Paris (FR)
EP 0 493 265 B1
Description
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The present invention relates to functional foods and beverages containing an algin such as alginic acid
or an alginate salt or ester which functions as dietary fibers. More particularly, the invention relates to algincontaining health foods and especially beverages, in which the algin has a decreased molecular weight
while still retaining its functions as a dietary fiber. Such algin-containing foods have a preventive effect on
obesity and diabetes.
Alginic acid is a high molecular weight, polyuronide-type polysaccharide which comprises D-mannuronic acid (abbreviated as M) and L-guluronic acid (abbreviated as G) combined in a varying proportion. It
is present between cells of brown algae. The molar ratio of M to G (M/G ratio) of alginic acid varies
depending on the species of the alga, season, and location in the algal body.
Alginic acid is made by extracting brown algae such as giant kelp (Macrocystis pyrifera) with an
aqueous sodium carbonate solution followed by precipitation with hydrochloric acid or calcium chloride.
Free alginic acid is very slightly soluble in water and tends to gel therein. Therefore, in practical use, a
water-soluble salt of alginic acid, e.g., an alginate salt with an alkali metal such as sodium is used in the
form of an aqueous solution. An aqueous solution of a water-soluble alginate salt is extremely viscous and
finds many applications, including as sizing agents, food additives, and paint thickeners.
The term "algin" used herein encompasses alginic acid and its derivatives including alginate salts and
esters.
Algin is known to have many desirable effects on human health. When an alginate salt is ingested, it is
converted into free alginic acid by a reaction with the acid in the stomach and gelled. The gelled alginic
acid cannot be absorbed by humans since mammals have no enzymes capable of digesting alginic acid.
Therefore, algin functions as a dietary fiber and is effective in intestinal regulation and prevention of
constipation. Sodium alginate is reported to be effective, when administered, in accelerating the excretion of
cholesterol and depressing the biological absorption of harmful metals such as strontium and cadmium. An
aqueous 5% solution of sodium alginate is clinically used as a drug in treatment of peptic ulcers and
esophagitis. Potassium and calcium alginates are reported to have a sodium-excreting activity [see, the
Japanese journal "Shokuhin Kaihatsu (Food Development)", Vol. 20, No. 3, pp. 20-23 (1985)].
In Japan, alginic acid, sodium alginate, and alginic acid propylene glycol ester are approved as food
additives, while in many European countries and the U.S.A., potassium alginate, ammonium alginate, and
calcium alginate are also approved. Thus, many species of algin have been proved to be harmless to the
human body.
On the basis of these advantageous effects of algin on health, a health beverage containing alginic acid
or sodium alginate has been proposed in Japanese Patent Application Laid-Open (Kokai) No. 55-28956(1980). Japanese Patent Application Laid-Open (Kokai) No. 1-240175(1989) discloses a high-fiber content
beverage which comprises water-insoluble dietary fibers dispersed in an aqueous dispersion medium
containing a gelling agent and a thickener. It is described therein that alginic acid and its derivatives can be
used as a gelling agent. This laid-open application teaches that algin can be merely used as a gelling agent
to thicken the beverage without the use of its function as a dietary fiber.
When an alginate salt such as sodium alginate is added to a beverage, it is not possible to increase its
content beyond a few percent, because a higher content causes the resulting solution to have an extremely
high viscosity which makes the solution no longer suitable for drinking. Therefore, an alginate salt can be
used only in the form of a dilute solution having a concentration of a few percent or lower even in
beverages as health foods. However, an algin-containing beverage having such a low content cannot
achieve the desirable effects of algin.
It is an object of the present invention to provide an algin-containing food, and particularly a health food
in the form of a beverage, which has a relatively high algin content and which can achieve the beneficial
effects of algin on health, and particularly its effects as a dietary fiber.
Another object of the invention is to provide a method for preparing such an algin-containing food.
As described above, a natural algin obtained by extraction of brown algae has a high molecular weight,
which is responsible for its relatively low solubility in water and the high viscosity of the resulting solution.
The present inventors attempted to decrease the molecular weight of a natural algin in order to increase its
water solubility and decrease the viscosity of the resulting solution.
It is known in the prior art that the molecular weight of a natural algin can be decreased by acid
hydrolysis or biodegradation with the aid of an enzyme. Acid hydrolysis involves an additional neutralization
step when the resulting lower molecular weight alginic acid is recovered. Biodegradation requires a long
time for the completion of degradation. Furthermore, both methods give an algin product having an
extremely low weight-average molecular weight on the order of less than 10,000. It cannot be expected that
3
EP 0 493 265 B1
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such a product still retains the desired functions of algin as a dietary fiber.
It has been found that heat treatment of a natural algin under pressure results in a decrease in weightaverage molecular weight in a controlled manner to a range of from 10,000 to 900,000, in which the
desirable effects of algin as a dietary fiber are still maintained. It has also been found that the resulting algin
having a decreased molecular weight is not only useful as a dietary fiber but is also effective for prevention
of obesity and diabetes.
The present invention provides an algin-containing food which comprises an algin having a weightaverage molecular weight in the range of 10,000 - 900,000.
The present invention also provides an algin-containing beverage for use as a health food which
comprises from 1% to 50% by weight of a water-soluble algin having a weight-average molecular weight in
the range of 10,000 - 900,000.
Such a low-molecular weight algin can be obtained from a high-molecular weight naturally-occurring
alginic acid or its derivative by heat treatment at 100 - 200 °C under pressure.
Figures 1(a) and 1(b) are graphs showing the effects of sodium alginate having an average molecular
weight of 10,000 on the levels of plasma glucose and insulin, respectively, upon glucose loading;
Figures 2(a) and 2(b) are graphs showing the effects of sodium alginate having an average molecular
weight of 50,000 on the levels of plasma glucose and insulin, respectively, upon glucose loading; and
Figures 3(a) and 3(b) are graphs showing the effects of sodium alginate having an average molecular
weight of 100,000 on the levels of plasma glucose and insulin, respectively, upon glucose loading.
The algin useful in the present invention has a weight-average molecular weight (hereinafter merely
referred to as average molecular weight) in the range of 10,000 - 900,000, which is much lower than that of
a conventionally used naturally-occurring alginic acid or its derivative. Such an algin can be prepared by
subjecting a naturally-occurring alginic acid or its derivative to heat treatment at a temperature of 100 200 °C under pressure for a time sufficient to decrease the molecular weight to 10,000 - 900,000 by thermal
degradation, although any algin can be used provided that it has an average molecular weight in the above
range. For the ease of drinking, the algin preferably has an average molecular weight of 10,000 - 150,000
and more preferably 50,000 - 100,000.
Theoretically, the algin may be any alginic acid or alginate salt which includes ammonium, alkali metal
(e.g., sodium and potassium), alkaline earth metal (e.g., magnesium and calcium), and other metal (e.g., iron
and tin) salts or alginate ester such as propylene glycol ester. However, since it is added to a beverage, it
must be selected from those algins which are approved as food additives. Therefore, useful algins are
limited, at present, to alginic acid and sodium, potassium, ammonium, and calcium salts and propylene
glycol ester of alginic acid.
In order to obtain the algin useful in the present invention, a naturally-occurring algin which is a highmolecular weight alginic acid or its derivative (alginate salt or ester) should be degraded to decrease the
average molecular weight to 10,000 - 900,000. The starting algin may be obtained from brown algae by
extraction in the method known in the art or it may be a commercially-available product. Those alginate
salts or esters which are not approved as food additives may also be used as a starting algin.
The degradation of the starting algin can be performed by subjecting the algin, preferably in the form of
an aqueous solution, to heat treatment at a temperature of 100 - 200 °C under pressure. At a temperature
below 100°C the desired degradation does not proceed at a satisfactory rate, while at a temperature above
200 ° C degradation proceeds excessively, resulting in the formation of an algin product having an average
molecular weight of less than 10,000, which loses its effects as a dietary fiber.
According to the book "Kaiso Kogyo (Alga Industry)", by Takeo Takahashi, published by Kogyo Tosho,
Japan, p. 235 (1941), alginic acid is subjected to heat treatment under pressure for the structural analysis
thereof. In the description in that book, the heat treatment was performed to obtain almost completely
degraded products of alginic acid which are useful for structural analysis and it was not intended to obtain a
low-molecular weight algin product which still retains its functions as a dietary fiber. Furthermore, there is
no description on the conditions for heat treatment.
The concentration of the aqueous solution to be treated is not critical and is usually in the range of
0.1% - 50% by weight. The pressure applied during heat treatment is preferably in the range of 0.1 - 15
kg/cm2-gauge. A suitable pressure reactor such as a high-temperature, high-pressure reactor or a conventional autoclave may be used to conduct the heat treatment depending on the pressure. The reactor may be
of the continuous, semi-continuous, or batch type and it may be either agitated or not agitated. The reaction
is continued until the starting algin is degraded to the desired average molecular weight. The reaction time
varies depending on the temperature, pressure, concentration, and other reaction conditions and is
generally in the range of from 1 minute to 100 hours.
4
EP 0 493 265 B1
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If necessary, the resulting low-molecular weight algin is converted into free alginic acid or an alginate
salt or ester which is approved as a food additive. The conversion can be performed by hydrolysis, ionexchange, neutralization, esterification, transesterification, or the like in a conventional manner. The resulting
aqueous solution may be concentrated or it may be converted into a powder by a suitable technique such
as concentration, freeze-drying, or spray drying.
Compared to the starting natural algin, the low-molecular algin product prepared in the above manner
and having an average molecular weight of 10,000 - 900,000 has a significantly increased solubility in water,
and the resulting aqueous solution has a significantly decreased viscosity at the same concentration. Still it
is effective as a dietary fiber. Therefore, it makes it possible to prepare a health food beverage having an
increased algin content. In contrast, with the starting natural algin, only a beverage having an algin content
of at most a few percent can be prepared and such a content may be insufficient to achieve the desirable
effects of algin.
The algin-containing food or beverage according to the present invention has an algin content in the
range of from 1% to 50% by weight. The algin content may be selected depending on the particular algin
and its molecular weight. The lower the molecular weight of the algin, the higher the maximum algin content
which can be used. In general, an algin content of less than 1% is not sufficient to be effective as a dietary
fiber, while an algin content of more than 50% causes the resulting aqueous solution to have an extremely
high viscosity, making the solution no longer suitable for drinking. Preferably the algin content is about 5% 20% by weight and more preferably about 5% - 10% by weight.
An algin-containing food can be prepared by adding the low-molecular weight algin in the form of either
a powder or an aqueous solution to an appropriate food. An algin-containing beverage can be prepared
either by dissolving the low-molecular weight algin in powder form in water or another drinkable liquid such
as fruit juice or by adjusting the concentration of the aqueous solution obtained by the above-described
heat treatment, if necessary.
If desired, the algin-containing food or beverage may further contain one or more food additives which
include sweeteners, flavors, preservatives, and coloring agents. In order to enhance the functions of the
beverage as a health food, one or more additives selected from other dietary fibers and various healthpromoting substances, e.g., iodine, iron-containing substance, fructo-oligosaccharide, and Lactobacillus
bifidus may be added to the beverage.
The algin-containing beverage contains an algin having a molecular weight which is high enough to
retain its effect as a dietary fiber and low enough to reduce the viscosity for ease of drinking, and it has a
high algin content sufficient to promote the health. Therefore, when drunk, the beverage can exert the
above-described favorable effects of algin on the health. Since an algin cannot be digested and hence is
noncaloric, an excess intake of an algin will not adversely affect the health to an appreciable degree.
The low molecular weight algin of the present invention is also useful in the prevention of obesity and
diabetes, particularly when it has an average molecular weight of 50,000 to 900,000. It has been proven that
such an algin suppresses an increase in blood glucose and insulin levels caused by glucose loading. It is
believed that this effect is the result of suppressing the absorption of glucose through the intestine or
increasing the metabolism and clearance of glucose in the peripheral tissues.
Therefore, the algin-containing food according to the present invention can effectively suppress an
abnormal increase in blood glucose and insulin levels caused by excessive intake of carbohydrates and
sugars derived from food. Thus, it is effective in reducing the activity of insulin to synthesize neutral fats
from glucose, thereby preventing obesity and diabetes. Suppression of an increase in blood glucose level is
also effective for prevention of aggravation of diabetes or prevention of obesity and hyperlipemia.
When the algin-containing food is used as a functional food for these purposes, it may be not only in
the form of a beverage as described above, but also in other forms such as a powder or jelly.
The number of patients suffering from diabetes accompanied by hyperlipemia or obesity is increasing
in Japan due to the change of diet toward a high fat diet from a high carbohydrate diet and due to an
increase in the intake of sugar. In the treatment of hyperlipemia and diabetes, a considerable number of
medicines have been developed and clinically used. In addition, functional or health foods such as dietary
fiber-containing beverages which are claimed to be effective for prevention of these diseases are sold on
the market. However, these foods rely upon the inhibition of cholesterol or triglyceride synthesis or
cholesterol absorption and result in a decrease in fat level.
In contrast, the low-molecular weight algin used in the present invention also affects the metabolism of
glucose. Therefore, it will be more effective for prevention of obesity and diabetes and prevent obesity and
aggravation of diabetes caused by excessive intake of glucose.
The following preparations, experiments, and examples are presented to further illustrate the present
invention.
5
EP 0 493 265 B1
Preparation of Low-Molecular Weight Algin
Preparations 1 - 7
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Commercially available sodium alginate (5 g) manufactured from giant kelp (Macrocystis pyrifera) and
95 g of water were mixed thoroughly and placed in an autoclave. The mixture was heat-treated in the
autoclave under the conditions (temperature and period) shown in Table 1 to thermally degrade the algin
under pressure. The pressure inside the autoclave was the autogenous pressure at the temperature used.
For example, the pressure was about 0.1 kg/cm2-gauge at 100°C and about 2 kg/cm2-gauge at 130°C. At
the end of the heat treatment, samples of the resulting aqueous solution containing a thermally-degraded
low molecular weight algin product were used to determine the weight-average molecular weight of the algin
product by GPC (gel permeation chromatography) and the viscosity of the solution at 30° C by a rotational
viscometer.
Preparation 8
Following the procedure used for the preceding preparations, 10 g of sodium alginate and 90 g of water
were subjected to thermal degradation under the same conditions as employed for Preparation 6.
20
Control
25
For comparison, the average molecular weight of the untreated sodium alginate used for the preceding
preparations and the solution viscosity thereof were determined using the starting mixture used for
Preparations 1 to 7.
The results obtained in these preparations and the control are also shown in Table 1.
Table
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1 -
Preparation
No.
Results
of
Heat
Treatment
Temperature
(°C)
of
Sodium
Period
(min)
Average
Alginate
MW
Viscosity
(cP)
1
130
60
220,000
108
2
120
60
900,000
975
3
110
60
1,400,000
8,000
4
100
60
1,800,000
14,800
5
130
90
140,000
6
130
120
60,000
7
130
250
10,000
1.5*
8
130
120
70,000
4.2"
Control
(Untreated)
2,700,000
30.5*
13*
26,500
measured
in an a q u e o u s
(Note)
was g e n e r a l l y
Viscosity
5.3%
at 30°C.
of
5
.
2
solution
at a c o n c e n t r a t i o n
* m e a s u r e d at 20°C;
** m e a s u r e d a t a c o n c e n t r a t i o n
of 10.7% a t 3 0 ° C .
The results of Table 1 demonstrate that sodium alginate having a molecular weight of 2,700,000 can be
degraded to form a low-molecular weight product having a molecular weight in the range of 10,000 1,800,000 by heat treatment under pressure. The higher the treatment temperature and the longer the
treating period, the lower the molecular weight of the product. Accordingly, it is apparent that the average
molecular weight of an algin can be lowered to a desired value in the range of 10,000 - 900,000 which is
suitable for beverages by appropriately selecting the conditions for heat treatment. The molecular weight
range of the degraded product was relatively narrow. Furthermore, the algin solutions obtained in Prepara6
EP 0 493 265 B1
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tions 1, 2, and 5 to 8 which had an average molecular weight of not greater than 900,000 in accordance with
the present invention had as low a viscosity as 1,000 cP or less. In contrast, the algin solutions obtained in
Preparations 3 and 4 which contained an algin having a molecular weight of 1,400,000 or 1,800,000 had
much higher viscosities of 8,000 cP and 14,800 cP, respectively, and were too viscous for drinking.
Effect of Low-Molecular Weight Algin on Intestinal Absorption of Harmful Substances
The following experiments were performed to illustrate that a low molecular weight algin obtained in the
above manner has an effect of suppressing the intestinal absorption of harmful substances to a degree
comparable to that of a conventional high molecular weight algin.
Test Procedure
is
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Male Wistar rats aged 8 weeks were used as test animals. Each test animal was given a radioactive
harmful substance in a predetermined dose, and immediately thereafter an aqueous solution of sodium
alginate was orally administered at a dose of 1.0 ml/100g-body weight. To the animals in a control group,
vehicle was administered in place of the sodium alginate solution. After the administration, urine and feces
of each animal were taken at regular intervals and the radioactivity thereof was measured to determine the
cumulative radioactivity expressed as a percent based on the radioactivity of the substance administered.
The % cumulative radioactivity was used as an indication for evaluating the effectiveness of the algin in
suppression of absorption of the harmful substance through the digestive tract. The harmful substances
used were cholesterol, trp-P-1 which is a carcinogen, and aflatoxin Bi which is a fungal toxin.
Experiment 1
An aqueous 1% solution of a low-molecular weight sodium alginate (abbreviated as AG-10, average
molecular weight: 100,000) and an aqueous 1% solution of an untreated sodium alginate (abbreviated as
AG-270, average molecular weight: 2,700,000) were orally administered immediately after the administration
of radioactive cholesterol and the % cumulative radioactivity in feces were determined to evaluate the effect
of the algin on absorption of cholesterol.
The results are shown in Table 2, from which it can be seen that the low molecular weight sodium
alginate significantly suppressed the absorption of cholesterol after 24 hours and its suppressive effect was
comparable to that of the untreated sodium alginate.
Experiment 2
The effect of the low-molecular weight and untreated sodium alginate on the intestinal absorption of trpP-1 was evaluated in the same manner as described in Experiment 1 except that radioactive trp-P-1 was
used as a harmful substance.
The results are shown in Table 3, from which it can be seen that the low molecular weight sodium
alginate significantly suppressed the absorption of trp-P-1 after 24 hours and its suppressive effect was
comparable to that of the untreated sodium alginate.
Experiment 3
The effect of the low-molecular weight and untreated sodium alginate on the intestinal absorption of
aflatoxin Bi was evaluated in the same manner as described in Experiment 1 except that radioactive
aflatoxin Bi was used as a harmful substance.
The results are shown in Table 4, from which it can be seen that the low molecular weight sodium
alginate significantly suppressed the absorption of aflatoxin Bi after 24 hours and its suppressive effect was
comparable to that of the untreated sodium alginate.
Experiment 4
55
Using aqueous 10% solutions of low molecular weight sodium alginates (AG-1, AG-5, and AG-10;
average molecular weight: 10,000, 50,000, and 100,000, respectively) and an untreated sodium alginate
(AG-270), the effects of these sodium alginates on intestinal absorption of cholesterol were evaluated in the
same manner as described in Experiment 1.
7
EP 0 493 265 B1
The results are shown in Table 5, from which it can be seen that the low molecular sodium alginate
having an average molecular weight of 50,000 and that having an average molecular weight of 100,000
significantly suppressed the absorption of cholesterol.
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Experiment 5
The effect of a low-molecular weight sodium alginate and untreated sodium alginate on the intestinal
absorption of trp-P-1 was evaluated in the same manner as described in Experiment 5 except that
radioactive trp-P-1 was used as a harmful substance and the radioactivity was measured of both urine and
feces.
The results are shown in Table 6 in terms of the % cumulative radioactivity of urine and feces. The
cumulative radioactivity of feces indicates that all the low molecular weight sodium alginates significantly
suppressed the absorption of trp-P-1 after 24 hours and that AG-5 was particularly effective. In contrast with
the results for feces, the cumulative radioactivity of urine in the administered groups was lower than that of
the control group. Thus, it was confirmed that these algins had an effect of suppressing the intestinal
absorption of trp-P-1 .
Experiment 6
20
25
The effect of a low-molecular weight sodium alginate and untreated sodium alginate on the intestinal
absorption of aflatoxin Bi was evaluated in the same manner as described in Experiment 5 except that
radioactive aflatoxin Bi was used as a harmful substance.
The results are shown in Table 7. The cumulative radioactivity of feces indicates that the low molecular
weight sodium alginates having an average molecular weight of 50,000 and 100,000 (AG-5 and AG-10)
significantly suppressed the absorption of aflatoxin Bi after 24 hours. In contrast with the results for feces,
the cumulative radioactivity of urine in the administered groups was lower than that of the control group.
Thus, it was confirmed that these algins had an effect of suppressing the intestinal absorption of aflatoxin
Bi.
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8
EP 0 493 265 B1
Table
2 -
Effect
Time
on
Intestinal
% Cumulative
Absorption
of
Cholesterol
Radioactivity1'
(hr )
Control
0-24
16.3+1.1
20.5+1.7*
17.1+1.0
-
48
24.1+0.9
31.9+2.4*
29.2+0.9*
-
72
30.9+1.6
37.3+2.2*
35.4+0.5*
-
96
35.5+1.8
41.9+2.0*
40.7+0.7*
AG-270
AG-10
1)
B a s e d on r a d i o a c t i v i t y
the f i g u r e s
administered;
are
mean v a l u e + s t a n d a r d
n=5;
error,
AG-270: U n t r e a t e d s o d i u m a l g i n a t e
(MW=2 , 700 , 000 ) ;
AG-10:
L o w - m o l e c u l a r weight sodium a l g i n a t e
(MW=100 , 000 ) ;
* Significance
of d i f f e r e n c e
(P<0.05)
between c o n t r o l
and
administered
t-test.
g r o u p s as d e t e r m i n e d w i t h S t u d e n t ' s
Table
3 -
Effect
Time
on
Intestinal
% Cumulative
Absorption
of
Trp-P-1
Radioactivity1'
(hr )
Control
0-24
22.2+4.8
30.9+2.3
29.5+0.1
-
48
32.2+1.6
36.5+1.7
35.0+0.9
-
72
33.6+1.0
37.0+1.6
36.1+1.0
-
96
33.9+1.1
37.5+1.7
36.4+0.9
1)
AG-270
AG-10
B a s e d on r a d i o a c t i v i t y
administered;
n=4-5
mean v a l u e + s t a n d a r d
error,
Table
4 -
Time
Effect
on
Intestinal
% Cumulative
Absorption
the
Aflatoxin
of
are
figures
BL
Radioactivity1'
(hr)
Control
0-24
20.7+2.2
25.9+4.7
20.7+1.1
-
48
26.2+1.9
29.3+3.7
30.9+1.5
-
72
31.4+0.4
32.6+3.1
33.3+1.0
-
96
31.9+0.5
33.3+3.1
33.7+1.8
1)
AG-10
AG-270
B a s e d on r a d i o a c t i v i t y
administered;
n=3-5
mean v a l u e + s t a n d a r d
error,
the
figures
are
EP 0 493 265 B1
Table
5 -
Effect
Time
on
Intestinal
Absorption
% Cumulative
of
Cholesterol
Radioactivity1
(hr )
Control
0 - 2 4
15.5+1.2
12.1+2.1
21.5+2.0*
24.8+1.3**
-
48
24.1+0.7
24.6+2.9
33.8+1.3**
34.2+0.6**
-
72
31.2+1.4
30.7+2.7
38.6+1.1**
40.2+0.7**
-
96
36.1+0.8
35.8+2.4
43.8+2.4**
45.9+0.5**
AG-1
AG-5
AG-10
1)
B a s e d on r a d i o a c t i v i t y
the f i g u r e s
administered;
are
n=3-6;
mean v a l u e + s t a n d a r d
error,
AG-1:
L o w - m o l e c u l a r weight sodium a l g i n a t e
(MW=10 , 000 ) ;
AG-5:
L o w - m o l e c u l a r weight sodium a l g i n a t e
(MW=50,000);
AG-10: L o w - m o l e c u l a r w e i g h t s o d i u m a l g i n a t e
(MW=100 , 000 ) ;
and
of d i f f e r e n c e
between c o n t r o l
*, **: S i g n i f i c a n c e
administered
t-test:
g r o u p s as d e t e r m i n e d w i t h S t u d e n t ' s
**=P<0.01
*=P<0.05,
Table
6 -
Effect
T ime
on
Intestinal
% Cumulative
(hr)
Control
Absorption
of
Trp-P-1
Radioactivity
AG-1
AG-5
AG-10
0-24
feces
urine
23.0+3.8
18.1+1.0
28.2+1.1
16.3+0.5
38.7+3.7*
15.5+0.4
32.2+1.6
16.2+0.5
-
48
feces
urine
32.9+1.5
20.6+1.1
36.4+0.2*
18.7+0.6
44.0+3.4**
17.6+0.4*
38.6+0.3*
18.7+0.6
-
72
feces
urine
34.1+1.0
22.2+1.2
37.1+0.3*
20.1+0.6
44.4+3.4**
19.2+0.3
39.4+0.9**
20.2+0.7
-
96
faces
urine
34.5+1.0
23.4+1.2
37.4+0.2*
21.0+0.6
45.6+3.3**
20.1+0.3*
40.1+0.3**
21.2+0.8
the f i g u r e s
B a s e d on r a d i o a c t i v i t y
are
administered;
n=3-5;
mean v a l u e + s t a n d a r d
error,
and
between c o n t r o l
of d i f f e r e n c e
*, **: S i g n i f i c a n c e
S
t
u
d
e
n
t
'
s
t-test:
w
i
t
h
administered
d
e
t
e
r
m
i
n
e
d
as
groups
**=P<0.01
*=P<0.05,
1)
10
EP 0 493 265 B1
7 -
Table
Effect
Intestinal
% Cumulative
Time
Control
(hr )
Absorption
of
Aflatoxin
Bt
Radioactivity1'
AG-1
AG-5
AG-10
0-24
feces
urine
21.0+2.5
14.2+1.8
19.3+1.3
13.3+0.5
22.4+4.9
13.7+1.3
25.5+1.8
13.0+1.1
-
48
feces
urine
26.6+2.3
22.0+2.8
29.9+1.2
20.2+0.8
34.7+0.9*
19.0+1.1
32.4+0.8*
18.5+0.7
-
72
feces
urine
31.5+0.5
27.6+3.2
32.8+1.0
24.0+1.6
36.5+1.1**
22.3+2.7
34.6+0.6**
22.2+0.7
-
96
faces
urine
32.0+0.5
30.8+3.6
33.8+1.8
26.2+1.0
37.1+0.7**
24.3+3.0
35.3+0.6**
24.3+1.2
10
15
on
the f i g u r e s
B a s e d on r a d i o a c t i v i t y
administered;
are
n=3-5;
mean v a l u e + s t a n d a r d
error,
and
between c o n t r o l
of d i f f e r e n c e
*, **: S i g n i f i c a n c e
t-test:
administered
g r o u p s as d e t e r m i n e d w i t h S t u d e n t ' s
**=P<0.01
*=P<0.05,
1)
20
25
Effect of Low-Molecular Weight Algin on Increase in Plasma Glucose and Insulin Levels in a Glucose
Tolerance Test
Experiment 7
30
35
40
Male Wistar rats having a body weight of 200 - 250 g (5 - 15 animals in each group) were
simultaneously orally administered 0.5 g per rat of glucose and 50 mg (1 ml as an aqueous 5% solution)
per rat of a low-molecular weight sodium alginate prepared as above using an oral sound. The animals in a
control group received glucose alone. In this experiment, sodium alginates having an average molecular
weight of 10,000, 50,000, and 100,000 (AG-1, AG-5, and AG-10) were used. Immediately and 10, 30, and 60
minutes after the administration, blood samples were collected by aparalytic cardiopuncture and were
centrifuged to separate the plasma. The plasma samples were assayed to determine the glucose and
insulin levels using Glucose B-Test assay kit (Wako-Junyaku Kogyo) and EIA INSULIN TEST-S assay kit
(Medicine and Biology Laboratories), respectively. The results are summarized in Tables 8 and 9 and
shown as graphs in Figures 1(a), 1(b), 2(a), 2(b), 3(a), and 3(b).
45
50
55
11
EP 0 493 265 B1
Table
8 -
Effect
on
Blood
Plasma
level
glucose
0 min
Group
10
Control
(n=15)
103.8+3.84
+ AG-1
(50 mg,
Glucose
Level
(mg/dl)
min
in
Glucose
[Mean
Tolerance
Value
30 min
60
+ SE]
min
145.3+5.07
140.5+4.61
147.2+7.24
110.2+11.4
n=5)
146.0+10.86
138.2+20.34
157.9+14.57
+ AG-5
(50 mg,
110.3+5.52
n=5)
137.8+7.86
111.1+6.02*
+ AG-10
(50 mg,
106.8+4.50
n=5)
147.6+6.69
114.8+9.66*
88.3+9.01**
134.6+10.15
AG-1:
L o w - m o l e c u l a r weight sodium a l g i n a t e
(MW=10,000);
AG-5:
L o w - m o l e c u l a r weight sodium a l g i n a t e
(MW=50,000);
AG-10: L o w - m o l e c u l a r w e i g h t s o d i u m a l g i n a t e
(MW=100 , 000 ) ;
of d i f f e r e n c e
between c o n t r o l
and
*, **: S i g n i f i c a n c e
administered
d
e
t
e
r
m
i
n
e
d
S
t
u
d
e
n
t
'
s
w
i
t
h
t-test:
g r o u p s as
**=P<0.01
*=P<0.05,
Table
9 -
Effect
on
Blood
Plasma
insulin
Insulin
level
Level
(/iIU/ml)
in
Glucose
[Mean
Tolerance
Value
Group
0 min
Control
(n»15)
19.6+1.91
43.1+6.77
31.1+5.19
22.8+2.34
+ AG-1
(50 mg,
20.2+4.02
n=5)
49.3+3.95
27.2+6.33
28.2+3.17
+ AG-5
(50 mg,
18.9+2.18
n=5)
57.7+8.82
30.9+7.99
18.4+3.46
+ AG-10
(50 mg,
19.1+3.40
n=5)
38.2+9.82
19.4+2.43*
23.7+2.54
10
30
min
min
60
+ SE]
min
and
*: S i g n i f i c a n c e
of d i f f e r e n c e
between c o n t r o l
administered
w
i
t
h
S
t
u
d
e
n
t
'
s
t-test:
d
e
t
e
r
m
i
n
e
d
as
groups
(P<0.05)
Sodium alginate having an average molecular weight of 50,000 (AG-5) significantly prevented the blood
glucose level from rising 30 and 60 minutes after the glucose loading or administration (P<0.05 and P<0.01 ,
respectively) as shown in Figure 2(a), but it had no effect on the blood insulin level as shown in Figure 2(b).
These results indicate that the algin, AG-5, not only may prevent the absorption of glucose through the
small intestine but also may promote the utilization of glucose in the peripheral tissues such as muscles.
As shown in Figure 3(a) and 3(b), sodium alginate having an average molecular weight of 100,000 (AG10) significantly prevented the blood glucose and insulin levels from rising 30 minutes after the glucose
loading (P<0.05). This indicates that AG-10 may prevent the absorption of glucose through the small
12
EP 0 493 265 B1
intestine.
Safety of Low Molecular Weight Algin
5
The following experiments illustrate that a low-molecular weight algin used in the invention is non-toxic
like a conventional high-molecular weight algin.
Experiment 8 (Acute Toxicity Test)
io
is
To each of 5-week old SD rats (28 male and 15 female rats), a single dose of 5 g/kg of a low-molecular
weight sodium alginate (average molecular weight: 50,000) was orally administered. The dose was about 25
times as much as that used in Experiment 7. Vehicle was given to the animals in a control group. After
administration, each rat was observed for 2 weeks with respect to its general condition and survival or death
with measurements of body weight and pathoanatomical observations, if necessary.
As is apparent from the results summarized below, no abnormalities were found.
(1) Observation of General Condition and Survival or Death
20
25
30
35
In all the male and female rats in the administered group, soft feces, sludgy feces, and soiling of pelage
around the ani with excrements were found within 4 hours after administration. However, these manifestations disappeared next day to restore normal conditions in all the animals.
In a female rat in the control group, soft feces were observed about 3 and a half hours after
administration, but these disappeared the next day.
Also in the control group, thinned pelage in the haunch were found in a female rat from the 1st to 6th
day after administration, and scabs in the neck (later resulting in thinned pelage) were found in a male rat
from the 11th day after administration. No further abnormalities in general condition were observed and
none of the rats died.
(2) Change in Body Weight
All the male and female rats in the administered group showed a change in body weight similar to the
control group.
(3) Pathoanatomical Observation
No abnormalities were found in either group.
Experiment 9 (Subacute Toxicity Test)
40
45
To each of 5-week old SD rats (60 male and 60 female rats), a low-molecular weight sodium
(average molecular weight: 50,000) was orally administered at a single daily dose of 1, 0.5, or 0.25
28 consecutive days. Vehicle was given to the animals in a control group. After administration, each
observed for 28 days with respect to its general condition and survival or death with measurements
weight and pathoanatomical observations, if necessary.
As is apparent from the results summarized below, no abnormalities were found.
alginate
g/kg for
rat was
of body
(1) Observation of General Condition and Survival or Death
50
55
In the male and female rats in the group administered a dose of 1 g/kg, soft feces were occasionally
found within 4 hours after administration but they disappeared next day.
Occasionally, thinned or fallen pelage in both the forelegs was occasionally found in all the groups
including the control group, and scabs in the neck were found in the control group and the group
administered a dose of 0.5 g/kg. No further abnormalities in general condition were observed and none of
the rats died.
(2) Change in Body Weight
In each administered group, the change in body weight was similar to that in the control group.
13
EP 0 493 265 B1
(3) Pathoanatomical Observation
No significant abnormalities were found in any group.
5
(Examples)
Using low-molecular weight sodium alginates having different average molecular weights, the following
beverage formulations for use as health foods were prepared by mixing the ingredients.
io
is
Example 1
100 g of aqueous 5.2% solution of sodium alginate (M.W.: 60,000)
5 g of sodium citrate
10 g of apple juice
5 g of honey
Example 2
20
25
30
35
200 g of aqueous 5.2% solution of sodium alginate (M.W.: 50,000)
5 g of sodium citrate
10 g of apple juice
5 g of honey
Example 3
200 g of aqueous 5.2% solution of sodium alginate (M.W.: 100,000)
5 g of sodium citrate
10 g of apple juice
5 g of honey
Example 4
200
5g
1g
5g
g of aqueous 5.2% solution of sodium alginate (M.W.: 50,000)
of sodium citrate
of ginseng extract
of royal jelly
Example 5
40
45
50
55
200 g of aqueous 5.2% solution of sodium alginate (M.W.: 100,000)
5 g of sodium citrate
10 g of extract of Houttuynia cordata
5 g of royal jelly
Example 6
200 g of aqueous 5.2% solution of sodium alginate (M.W.: 100,000)
5 g of sodium citrate
10 g of adlay extract
5 g of honey
All the resulting beverages were easy to drink and had a good taste. Thus, the addition of a lowmolecular weight algin which still functions as a dietary fiber to a beverage or food according to the present
invention can provide a health food having a higher algin content than that prepared from a conventional
high-molecular weight algin. Since the low-molecular weight algin can suppress an increase in blood
glucose or insulin level in a glucose tolerance test, the algin-containing food according to the invention will
be useful as a functional food for prevention of obesity and diabetes.
14
EP 0 493 265 B1
Claims
5
10
1.
An algin-containing food which comprises an algin having a weight-average molecular weight in the
range of 10,000 - 900,000.
2.
The algin-containing food of Claim 1, wherein the algin has a weight-average molecular weight in the
range of 10,000 - 150,000.
3.
The algin-containing food of Claim 1, wherein the algin content is from 1% to 50% by weight.
4.
The algin-containing food of Claim 3, wherein the algin content is from 5% to 20% by weight.
5.
The algin-containing food of Claim 1, wherein the algin is prepared by subjecting a high-molecular
weight naturally occurring algin or its derivative to heat treatment at a temperature of 100 - 200 °C
under pressure for a time sufficient to decrease its weight-average molecular weight to 10,000 900,000.
6.
The algin-containing food of Claim 1 in the form of a powder or jelly.
7.
An algin-containing health food beverage comprising from 1% to 50% by weight of a water-soluble
algin having a weight-average molecular weight in the range of 10,000 - 900,000.
8.
The algin-containing beverage of Claim 7, wherein the algin has a weight-average molecular weight in
the range of 10,000 - 150,000.
9.
The algin-containing beverage of Claim 7, wherein the algin content is from 5% to 20% by weight.
is
20
25
30
10. The algin-containing beverage of Claim 7, wherein the algin is prepared by subjecting a high-molecular
weight naturally occurring algin or its derivative to heat treatment at a temperature of 100 - 200 °C
under pressure for a time sufficient to decrease its weight-average molecular weight to 10,000 900,000.
Patentanspruche
1.
Algin enthaltendes Nahrungsmittel, welches ein Algin mit einem gewichtsmittleren Molekulargewicht im
Bereich von 10000 bis 900000 umfaBt.
2.
Algin enthaltendes Nahrungsmittel nach Anspruch 1, wobei das Algin ein gewichtsmittleres Molekulargewicht im Bereich von 10000 bis 150000 besitzt.
3.
Algin enthaltendes Nahrungsmittel nach Anspruch 1, wobei der Algingehalt 1 bis 50 Gew.-% betragt.
4.
Algin enthaltendes Nahrungsmittel nach Anspruch 3, wobei der Algingehalt 5 bis 20 Gew.-% betragt.
45
5.
Algin enthaltendes Nahrungsmittel nach Anspruch 1, wobei das Algin hergestellt wird, indem ein
hochmolekulargewichtiges, naturlich vorkommendes Algin oder ein Derivat davon einer Warmebehandlung bei einer Temperatur bei 100 - 200 °C unter Druck uber einen ausreichenden Zeitraum unterzogen
wird, urn dessen gewichtsmittleres Molekulargewicht auf 10000 - 900000 zu verringern.
50
6.
Algin enthaltendes Nahrungsmittel nach Anspruch 1 in Form eines Pulvers oder Gelees.
7.
Algin enthaltendes Gesundheitsnahrungsmittel-Getrank, umfassend 1 bis 50 Gew.-% eines wasserloslichen Algins mit einem gewichtsmittleren Molekulargewicht im Bereich von 10000 - 900000.
8.
Algin enthaltendes Getrank nach Anspruch 7, wobei das Algin ein gewichtsmittleres Molekulargewicht
im Bereich von 10000 - 150000 besitzt.
9.
Algin enthaltendes Getrank nach Anspruch 7, wobei der Algingehalt 5 bis 20 Gew.-% betragt.
35
40
55
15
EP 0 493 265 B1
5
10. Algin enthaltendes Getrank nach Anspruch 7, wobei das Algin hergestellt wird, indem ein hochmolekulargewichtiges, naturlich vorkommendes Algin oder ein Derivat davon einer Warmebehandlung bei einer
Temperatur von 100 - 200 °C unter Druck uber einen ausreichenden Zeitraum unterzogen wird, urn
dessen gewichtsmittleres Molekulargewicht auf 10000 - 900000 zu reduzieren.
Revendicatlons
1.
Aliment contenant de I'algine qui comprend une algine ayant une masse moleculaire moyenne en poids
comprise entre 10.000 et 900.000.
2.
Aliment contenant de I'algine selon la revendication 1, dans lequel I'algine presente une masse
moleculaire moyenne en poids comprise entre 10.000 et 150.000.
3.
Aliment contenant de I'algine selon la revendication 1, dont la teneur en algine est de 1 a 50% en
poids.
4.
Aliment contenant de I'algine selon la revendication 3, dans lequel la teneur en algine est de 5 a 20%
en poids.
20
5.
Aliment contenant de I'algine selon la revendication 1, dans lequel on prepare I'algine en soumettant
une algine naturelle de haute masse moleculaire ou son derive a un traitement thermique a une
temperature de 100 a 200 °C sous pression pendant une duree suffisante pour abaisser sa masse
moleculaire moyenne en poids a une valeur de 10.000 a 900.000.
25
6.
Aliment contenant de I'algine selon la revendication 1 sous forme d'une poudre ou d'une gelee.
7.
Boisson contenant de I'algine a usage d'aliment pour la sante qui comprend de 1 a 50% en poids
d'une algine hydrosoluble ayant une masse moleculaire moyenne en poids de 10.000 a 900.000.
8.
Boisson contenant de I'algine selon la revendication 7, dans lequel I'algine presente une masse
moleculaire moyenne en poids de 10.000 a 150.000.
9.
Boisson contenant de I'algine selon la revendication 7, dans laquelle la teneur en algine est de 5 a 20%
en poids.
10
is
30
35
40
10. Boisson contenant de I'algine selon la revendication 7, dans laquelle on prepare I'algine en soumettant
une algine naturelle de haute masse moleculaire ou son derive a un traitement thermique a une
temperature de 100 a 200 °C sous pression pendant une duree suffisante pour abaisser sa masse
moleculaire moyenne en poids a une valeur de 10.000 a 900.000.
45
50
55
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